A striking conclusion from single-gene deletion studies in S. cerevisiae is that ~80% of genes are non- essential in rich media. Of these, ~84% are non-essential in all of 21 diverse growth conditions. Double mutant studies confirm that gene loss is often buffered by the presence of other genes with compensatory function. Thus, mutations in multiple genes are often required to reveal gene function. Where a large gene family with overlapping function exists, one must delete many genes to eliminate the function. For example, at least 20 genes must be deleted to eliminate glucose transport. In another example, there is substantial overlapping function among the 29 ABC transporter homologs (drug efflux pumps) in yeast. Multi-mutant strains deficient in a function allow cloning of orthologous genes by functional complementation, or 'add-back' experiments allowing study of single genes in isolation. Here we propose the "Green Monster" method, a high risk / high reward technology for rapidly engineering strains carrying many precise deletions: 1) for each target gene, a "ProMonster" strain is constructed, carrying a precise replacement of one of the target genes with GFP under an inducible Tet promoter; 2) ProMonster strains are pooled and repeatedly mated en masse with one another, sporulated; 3) increasing GFP gene dosage is selected by fluorescence-activated cell sorting (FACS), thereby selecting strains with more deletions. The number of mutant alleles is expected to double in each early round, and continue to grow quickly. Simulations show that a strain carrying 24 deletions would require as few as 8 rounds of mating, sporulation, and FACS selection. Resulting Green Monster strains can be typed using locus- specific PCR primers or 'bar-code' microarrays. This strategy can also be applied to alleles of other types-e.g., inducible promoter alleles allowing synchronous change in expression of all members of a protein complex, or insertion of many exogenous genes (allowing study of complex mammalian pathways in a more tractable genetic system). A potential future extension is a 'genetic pull-down' technique for discovering sets of genes that exhibit masking epistasis or suppression of a mutation in a given query gene. Many genes of high relevance to public health--e.g., those encoding proteins that provide drug resistance to pathogens and tumors by exporting drugs from the cell--are members of large families of related genes, while other disease-relevant genes act in concert with other genes to encode protein complexes. We propose methods for efficiently engineering yeast strains in which many selected genes have been precisely deleted or inserted from another organism. Multiply-deleted strains allow functional study of genes in gene families, while multiply-inserted strains allow the study of human protein complexes in a tractable model organism. [unreadable] [unreadable] [unreadable]